The CFTR chloride channel is implicated in two major human diseases: cystic fibrosis (low CFTR activity) and secretory diarrhea (excessive CFTR activity). The development of rational treatment strategies for either disease requires a better understanding of what activates or inactivates the CFTR channel. Although it is clear that CFTR is stimulated by PKA-mediate phosphorylation of the large regulatory domain (R domain) within this channel, the mechanisms that control CFTR gating are still obscure. Two new paradigms of CFTR regulation will be explored: 1) the stabilization of CFTR channel activity by an intramolecular interaction between the amino-terminal tail (N-tail) and the R domain and 2) the coupling of CFTR gating to the membrane traffic machinery by an intermolecular interaction between the CFTR N-tail and syntaxin 1A. These paradigms will be explored by pursuing three Specific Aims. First, the mechanism by which the N-tail stabilizes CFTR channel activity will be defined. Subaims include testing the hypothesis that the N-tail controls channel gating by modulating the phosphorylation of key residues within the R domain. Second, the structural basis of the interaction between the N-tail and R domain will be defined. The nature of the physical interaction between these domains will be characterized and the hypothesis that CFTR channel gating can be disrupted by peptides that block this interdomain interaction will be tested. Third, the hypothesis that CFTR channel gating is regulated by interactions between this ion channel will be tested and that components of the membrane traffic machinery coordinate the regulation of ion transport and protein traffic in epithelial cells. The results of the proposed study should provide new information regarding the mechanisms that control the activity of the CFTR chloride channel; information that may lead to more effective strategies for manipulating CFTR function in diseases that involve this ion channel.